氟苯尼考致小鼠L细胞毒副作用的线粒体途径的研究

发布时间：2018-04-05 08:20

  本文选题：氟苯尼考　切入点：毒副作用　出处：《山东农业大学》2017年博士论文

【摘要】：随着我国畜牧养殖规模的不断扩大,畜禽疫病防控的压力日益增大,多病原混合感染特别是细菌继发感染现象较为普遍,大量抗生素被广泛应用于畜禽疾病的预防及兽医临床治疗,并在饲料中被大量添加以提高饲料转化率或达到促生长的目的。在此背景下,畜禽生产中滥用和乱用抗菌药物的现象十分普遍,这给人畜健康和细菌病的防控带来了巨大挑战。氟苯尼考(FLO)是一种兽医临床专用的新型广谱抗菌药物,目前已成为畜牧及水产养殖过程中使用范围最广、使用量最大的抗菌药物之一。“内共生学说”指出真核生物的线粒体起源于古细菌,古细菌寄生于原始生物后随着长时间的互利共生与演化过程而演变成了细胞线粒体,因此线粒体核糖体在结构和功能上与细菌核糖体具有较高的相似性。基于此,真核细胞线粒体的核糖体便很容易受到特定抗菌药物的干扰,并给动物机体或细胞带来严重的毒副作用。FLO能够与细菌核糖体A位点紧密结合而抑制肽酰转移酶的活性,最终通过影响蛋白质的合成而杀灭细菌,同时也会影响真核细胞线粒体蛋白质的合成。随着FLO的大量应用,关于其毒副作用尤其是造血免疫毒性和胚胎毒性的报道也越来越多,但都基于FLO对机体生理机能或组织器官功能的影响观察或检测,尚未见到在细胞分子水平上研究FLO毒副作用的报道。为明确FLO对线粒体结构与功能的具体影响,及其对细胞存活、细胞增殖及细胞稳态的毒副作用,本研究以成纤维细胞为研究对象,在细胞及分子水平上研究了FLO对线粒体结构与功能的影响,以及线粒体损伤对细胞增殖、细胞存活及线粒体自噬的影响及其机制,从分子水平揭示了FLO致成纤维细胞毒性的具体机制。一、FLO诱导线粒体结构与功能损伤线粒体作为真核细胞内进行有氧呼吸的主要场所,其结构和功能的完整对细胞的生命活动来说至关重要。本研究首先聚焦在FLO对线粒体结构与功能的影响方面,选用不同剂量的FLO(0.4、0.1、0.025 mg/m L)处理细胞48 h后,主要进行以下实验:(1)Western blot分别检测FLO对线粒体核糖体编码的蛋白质Cox I及细胞质核糖体编码的蛋白质白Cox IV的表达水平的影响;(2)透射电镜检查FLO对细胞内线粒体数量和形态结构的影响;(3)流式细胞术分别检测FLO对线粒体膜电位及细胞内活性氧簇(ROS)生成量的影响;(4)生化方法分别测定FLO对线粒体呼吸链复合体I、II及IV的催化活性及细胞内ATP生成量的影响。结果表明:FLO能够显著抑制由线粒体核糖体翻译的线粒体蛋白如Cox I的表达,进而显著下调线粒体呼吸链复合体I及IV的活性(p0.01)、显著降低线粒体膜电位(p0.05或p0.01)及胞内ATP的水平(p0.05或p0.01)并提高ROS的生成量;从结构上来说,FLO处理后的细胞内出现了肿胀、空泡化、嵴消失的损伤线粒体。以上结果表明线粒体正常的结构和生理功能受到了FLO的严重影响。二、FLO诱导的线粒体功能障碍抑制细胞增殖活性正常线粒体在细胞周期调控和细胞增殖发育等方面具有重要作用,鉴于FLO可以造成明显的线粒体功能障碍,我们随后从以下几方面研究了FLO对细胞存活、细胞周期和增殖调控的影响:(1)不同剂量FLO分别处理细胞12 h、24 h、36 h、48 h、60 h及72 h,通过细胞计数研究细胞增殖曲线;(2)CCK-8还原法测定不同剂量FLO处理细胞24 h、48 h及72 h对细胞增殖活性的影响;(3)Ed U掺入实验检测不同剂量FLO处理48 h对细胞DNA复制活性的影响;(4)流式细胞术检测不同剂量FLO对细胞周期分布的影响;(5)流式细胞术及caspase-3活性分析实验检测FLO对细胞凋亡的影响;(6)乳酸脱氢酶(LDH)释放实验检测FLO对细胞死亡率的影响;(7)Western blot检测FLO对细胞内增殖相关信号通路活化状态的影响。结果表明:(1)FLO能导致与剂量有关的细胞活力降低,表现在细胞绝对数量的下降和增殖活力的降低(p0.01);(2)Ed U掺入试验及细胞周期检测结果表明FLO能显著降低发生DNA复制的细胞比例,并使细胞周期发生G0/G1期阻滞(p0.05或p0.01);(3)选用D-半乳糖培养基迫使细胞只能通过线粒体呼吸链供能以便于使线粒体毒性物质对线粒体的毒副作用表现得更为明显,给予FLO后发现多种细胞表现出了更为严重的细胞活力抑制作用(p0.01),表明FLO对线粒体呼吸链的损伤与其所致的细胞活力下降直接相关;(4)FLO对细胞凋亡率和坏死率无明显影响(p0.05),说明FLO所致细胞活力下降仅与细胞增殖减缓有关,与细胞死亡则无明显关联;(5)从机制上来说,FLO活化了AMPK-m TOR-p70S6K通路,即FLO使活化的AMPK(磷酸化)表达上调、磷酸化的m TOR及磷酸化的p70S6K表达下调,同时ROS-p53-p21通路也被活化,AMPK特异性抑制剂Compound C和抗氧化剂NAC分别能够特异性抑制上述相应通路的活化并提高被FLO抑制的细胞增殖活性(p0.05或p0.01)。三、FLO导致细胞内损伤线粒体清除障碍及细胞衰老发生线粒体自噬对损伤线粒体的清除和细胞稳态的维持至关重要,鉴于FLO可以造成明显的线粒体功能障碍,我们随后从以下几方面研究了FLO对细胞内损伤线粒体的清除过程即线粒体自噬的影响:(1)Western blot检测FLO对胞内线粒体质量(线粒体内膜蛋白Tim23水平)的影响;(2)使用线粒体特异性探针Mito Tracker Green染色线粒体,并用流式细胞术检测FLO对细胞内线粒体数量的影响;(3)使用自噬相关蛋白LC3的特异性抗体染色结合共聚焦显微镜检查FLO处理的细胞内自噬点的形成情况;(4)Western blot检测FLO对自噬相关蛋白LC3B II/I及p62的表达水平,以评估细胞自噬水平;(5)衰老相关β-半乳糖苷酶染色检测FLO处理8 d所造成的的损伤线粒体累积对细胞衰老水平的影响;(6)Western blot检测FLO对招募至损伤线粒体的线粒体自噬起始关键蛋白Parkin及p62水平的影响,结合ROS清除剂NAC研究p53蛋白对Parkin向线粒体转位的影响;(7)Mito Tracker Green染色结合流式细胞术检测NAC对线粒体自噬或损伤线粒体清除功能的调节作用。结果表明:(1)细胞自噬功能在FLO处理的细胞中处于抑制状态,表现在自噬相关蛋白LC3B II下降和p62蛋白表达水平上升,细胞内LC3荧光聚点的数量在不同处理组间无显著差异;(2)胞内线粒体内膜蛋白Tim23的水平在FLO给药后轻微升高,FLO处理组的Mitotracker Green荧光值显著升高,表明FLO处理后损伤的线粒体并不能通过自噬有效清除,而是堆积在细胞内;(3)Parkin蛋白从胞浆被招募到线粒体是线粒体自噬的关键环节,本研究发现FLO处理后线粒体膜上Parkin蛋白的量及其招募的p62蛋白的量都未升高,表明FLO所致线粒体自噬异常与Parkin蛋白的线粒体转位障碍有关;(4)使用NAC和FLO共处理细胞后发现Parkin蛋白线粒体转运障碍能够得到缓解,结合先前p53蛋白抑制Parkin蛋白线粒体转位的报道可知,FLO处理活化了ROS-p53通路并通过抑制Parkin蛋白的线粒体转位发挥线粒体自噬抑制效应,进而使损伤的线粒体得不到有效清除并最终导致细胞衰老的发生。本研究从分子水平揭示了氟苯尼考致成纤维细胞毒性的具体机制,研究结果对于明确线粒体毒性药物的毒性机制具有重要借鉴意义,有助于指导氟苯尼考在畜牧及水产养殖中的合理应用;同时对靶向线粒体的抗肿瘤药物研发及新一代抗生素的线粒体安全性评价具有借鉴意义。
[Abstract]:Along with our country animal husbandry continues to expand the scale of animal disease prevention and control, increasing pressure, multiple pathogens infection especially secondary bacterial infection is a common phenomenon, a large number of antibiotics have been widely used in livestock and poultry disease prevention and veterinary clinical treatment, and in the feed was added in order to improve the feed conversion rate reached the purpose of promoting the growth or in this context, livestock and poultry production in the abuse and misuse of antibiotics is a common phenomenon, which is a great challenge to human and animal health and disease prevention and control bacteria. Florfenicol (FLO) is a new kind of broad-spectrum antibiotics for Veterinary Clinical College, has become the most widely used animal husbandry and aquaculture in the process, one of the largest amount of antimicrobial drug use. "The endosymbiotic theory" that the mitochondrial origin of eukaryotes from ancient bacteria, archaea parasitic in primitive organisms with long interaction The symbiosis and evolution and evolved into mitochondria, mitochondria ribosomes in the structure and function of the bacterial ribosome has high similarity. Based on this, the eukaryotic cell mitochondrial ribosomes is easily interfered by certain antibiotics, and to animal body or cell toxicity can be combined with the severity of.FLO closely with the bacterial ribosome A sites and inhibit peptidyl transferase activity by affecting protein synthesis and eventually kill bacteria, but also affect the synthesis of eukaryotic mitochondrial proteins. With a large amount of FLO should be used, especially on the toxicity and embryo toxicity hematopoietic immune toxicity reported more and more, but on the basis of FLO on organ function or tissue physiological effects observed or detected, we haven't seen FLO toxicity at the molecular level is clearly F reported. The effect of LO on mitochondrial structure and function, and on cell survival, cell proliferation and cell homeostasis toxicity in this study fibroblasts as the research object, at the cellular and molecular level study on the effect of FLO on the structure and function of mitochondria, and mitochondrial damage on cell proliferation, cell viability and mitochondrial effects autophagy and its mechanism, reveals the specific mechanism of FLO induced into fiber cell toxicity at the molecular level. First, FLO induced mitochondrial mitochondrial structure and function damage in eukaryotic cells as the main place for aerobic respiration, for cellular activity the integrity of its structure and function is very important. This paper focused on FLO effect of mitochondrial structure and function, with different doses of FLO (0.4,0.1,0.025 mg/m L) treated cells after 48 h, mainly for the following experiments: (1) Western blot respectively. The expression level of FLO on the measurement of mitochondrial ribosomal protein Cox encoding I encoding protein and cytoplasmic ribosomes Cox IV; (2) influence the transmission electron microscopy FLO on the number and structure of mitochondria in cells; (3) were detected FLO on ROS mitochondrial membrane potential and intracellular flow cytometry. Surgery (ROS) effect of production; (4) were determined by FLO on mitochondrial respiratory chain complex I biochemical method, influence the production of ATP catalytic activity and cell II and IV. The results show that FLO can significantly inhibit the mitochondrial ribosomal translation of mitochondrial proteins such as Cox and I expression, significantly decreased mitochondrial respiration the chain complex I and IV activity (P0.01), significantly decreased the mitochondrial membrane potential (P0.05 or P0.01) and intracellular levels of ATP (P0.05 or P0.01) and increase the production of ROS; from the structure, at FLO after cell swelling, Vacuolization, cristae disappeared. These results showed that the damage of structure and physiological function of mitochondria was severely affected by FLO. Two, mitochondrial dysfunction induced by FLO inhibition of cell proliferation activity of normal mitochondria plays an important role in the regulation of cell cycle and cell proliferation, development and other aspects, in view of the fact that FLO can cause mitochondrial dysfunction obviously, then we from the following aspects of the FLO effect on cell survival, cell cycle and proliferation regulation: (1) were treated with different doses of FLO cells in 12 h, 24 h, 36 h, 48 h, 60 h and 72 h, the cell count of cell proliferation curve; (2) the determination of different dose of FLO 24 cells h CCK-8 reduction method, 48 h and 72 h on the cell proliferation effect; (3) Ed U incorporation assay with different doses of FLO treatment for 48 h replication activity of cell DNA; (4) different doses of FLO were detected by flow cytometry The distribution of cell cycle; (5) flow cytometry and caspase-3 assay. FLO activity analysis on cell apoptosis; (6) lactate dehydrogenase (LDH) release assay of FLO on cell death rate; (7) the activation of Western blot detection of FLO on cellular proliferation related signaling pathways influence. The results showed that: (1) FLO can lead to dose related reduction of cell viability, reflected in the decline in the absolute number of cells and the proliferation activity decreased (P0.01); (2) Ed U incorporation assay and cell cycle assay showed that FLO can significantly reduce the occurrence of DNA replication and the proportion of cells in the cell cycle of G0/G1 block (P0.05 or P0.01); (3) using D- galactose medium forcing cells only through the mitochondrial respiratory chain of energy supply in order to facilitate the mitochondrial toxicity of substances on mitochondrial toxicity is more obvious, after giving FLO found a variety of cell surface A more serious inhibition of cell viability (P0.01), showed that FLO damage on the mitochondrial respiratory chain and cellviability decreased by direct correlation; (4) FLO on cell apoptosis rate and necrosis rate had no significant effect (P0.05), indicating that FLO induced cell viability decreased only related to cell proliferation is slow, no significant correlation with cell death; (5) from the mechanism, FLO activated AMPK-m TOR-p70S6K pathway, namely FLO activated AMPK (phosphorylated) expression and phosphorylation of M TOR and phosphorylated p70S6K expression, while ROS-p53-p21 pathway was also activated, AMPK specific inhibitor Compound C and antioxidant NAC which can activate specific inhibition of the corresponding pathway and improve the cell proliferation activity was inhibited FLO (P0.05 or P0.01). Three FLO, leading to cell damage within the mitochondria and remove obstacles and senescence occurs on mitochondrial autophagy damage line Critical particle clearance and maintain cellular homeostasis, whereas FLO can cause mitochondrial dysfunction obviously, we then from the following aspects of removing FLO on cell injury of mitochondria that mitochondrial autophagy: (1) Western blot detection of FLO on the intracellular mitochondrial mass (mitochondrial protein Tim23) effect; (2) mitochondrial staining using a mitochondria specific probe Mito Tracker Green, and flow cytometry was used to detect the number of intracellular FLO on mitochondrial effects; (3) specific antibodies using autophagy related protein LC3 staining combined with confocal microscopy formation of FLO cells in autophagy point; (4) expression the level of Western blot FLO on autophagy related protein LC3B II/I and p62, to evaluate the level of autophagy; (5) senescence associated beta galactosidase staining to detect the FLO 8 D treatment caused by the The cumulative effect of mitochondrial damage on cell senescence level; (6) effects of Western blot FLO on the detection of mitochondrial damage to recruit mitochondrial autophagy protein Parkin start key and the level of p62, combined with the ROS scavenger NAC study of p53 protein effect on Parkin translocation to the mitochondria; (7) Mito Tracker Green staining combined with flow cytometry NAC to remove or damage the mitochondrial mitochondrial autophagy function. The results showed that: (1) the function of autophagy is inhibited in FLO treated cells, expressed in autophagy related protein LC3B decreased II and p62 protein expression level increased, no significant difference in the number of intracellular LC3 fluorescence accumulation in different treatment groups (; 2) intracellular mitochondrial membrane protein Tim23 levels increased slightly after FLO administration, Mitotracker Green fluorescent FLO treatment group was significantly increased after FLO treatment showed that mitochondria do not damage Through autophagy effectively cleared, but the accumulation in the cells; (3) Parkin protein from the cytoplasm is recruited to mitochondria is a key link of mitophagy, this study found that after the treatment of FLO mitochondrial Parkin protein and p62 protein recruitment amount did not increase, suggesting mitochondrial translocation and Parkin protein abnormal disorder FLO induced mitochondrial autophagy; (4) using NAC and FLO cells treated with Parkin protein found in mitochondrial disorders can be mitigated, combined with the previous p53 protein Parkin protein inhibiting mitochondrial translocation reports shows that FLO activation of the ROS-p53 pathway and the mitochondrial translocation of Parkin protein inhibits mitochondrial autophagy play inhibitory effect, and then make the injury mitochondria not effectively remove and eventually lead to cell senescence. The research from the molecular level reveals the florfenicol induced fibroblast cell toxicity The specific mechanism, the research results have important significance for the mechanism of toxicity of clear mitochondrial toxicity of drugs, helps to guide the rational application of florfenicol in animal husbandry and aquaculture; at the same time, mitochondria targeted anticancer drug development and a new generation of antibiotics in the mitochondrial safety evaluation has the reference significance.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S859.82

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